A series of azido pyridine and adenine nucleotide photoaffinity analogues have been developed in this laboratory as general reagents for the investigation of the nucleotide binding regions of proteins. These compounds have been shown to act as competitive inhibitors of alcohol dehydrogenase and NADH-CoQ reductase (pyridine nucleotide analogues) and myosin and F1 (mitochondrial) ATPase (adenine nucleotide analogues) and following illumination to be irreversible inhibitors. The irreversible inhibition has been found to be associated with a covalent labeling of the proteins via the photogeneration of a reactive nitrene in the case of the myosin and F1 (ATPase) and presumably is responsible for the photodependent inhibition of NADH-CoQ reductase by arylazido pyridine nucleotides. We propose to combine the use of the resolving power of sodium dodecylsulfate and urea preparatory gel electrophoresis with photoaffinity labeling techniques to isolate and characterize the individual protein components of the NADH-CoQ reductase of ox-heart mitochondria. The covalent binding of radio labeled nucleotide analogues will be used to delineate the specific proteins or subunits responsible for NADH dehydrogenase activity as well as other enzymatic activities associated with Complex I. The study will aid in the characterization and interpretation of the mechanism of mitochondrial electron transport as well as the various dehydrogenase activities of this multi enzyme complex.